Analysis of retinoic acid receptor beta expression in normal and malignant laryngeal mucosa by a sensitive and routine applicable reverse transcription-polymerase chain reaction enzyme-linked immunosorbent assay method.

Abstract

Retinoic acid receptor beta (RAR-beta) seems to be a useful intermediate marker in trials of retinoids, and the aim of this work was to describe a fast, sensitive, and routine applicable method to measure RAR-beta expression in tumor biopsies. We developed a new technique combining reverse transcription-PCR with a colorimetric ELISA detection of amplification products. The principle of this nonradioactive method is based on digoxigenin labeling of PCR products during amplification. Amplified DNA is hybridized with a biotinylated capture probe. The generated hybrid is immobilized on a streptavidin-coated microtiter plate, and detection is performed with the use of an antidigoxigenin peroxidase conjugate. We applied this method to quantify the expression of RAR-beta and an internal control (beta2 microglobulin) in laryngeal tumors. We found a detection threshold at 50 pg of PCR products, which represents a 100-fold improvement when compared to the detection limit of ethidium bromide detection. The method was reproducible (intra- and interassay reproducibilities at 7 and 5%, respectively). We used this technique for determining RAR-beta expression in 20 patients with laryngeal carcinoma and in 20 patients without cancer. The data show that the value of the RAR-beta:beta2 microglobulin ratio is decreased in tumoral versus nontumoral specimens (P = 0.0012), which is consistent with previously published results.